Abstract
Gaucher disease (GD) is caused by deficiency of the lysosomal membrane enzyme glucocerebrosidase (GCase) and the subsequent accumulation of its substrate, glucosylceramide (GC). Mostly missense mutations of the glucocerebrosidase gene (GBA) cause GCase misfolding and inhibition of proper lysosomal trafficking. The accumulated GC leads to lysosomal dysfunction and impairs the autophagy pathway. GD types 2 and 3 (GD2-3), or the neuronopathic forms, affect not only the Central Nervous System (CNS) but also have severe systemic involvement and progressive bone disease. Enzyme replacement therapy (ERT) successfully treats the hematologic manifestations; however, due to the lack of equal distribution of the recombinant enzyme in different organs, it has no direct impact on the nervous system and has minimal effect on bone involvement. Small molecules have the potential for better tissue distribution. Ambroxol (AMB) is a pharmacologic chaperone that partially recovers the mutated GCase activity and crosses the blood-brain barrier. Eliglustat (EGT) works by inhibiting UDP-glucosylceramide synthase, an enzyme that catalyzes GC biosynthesis, reducing GC influx load into the lysosome. Substrate reduction therapy (SRT) using EGT is associated with improvement in GD bone marrow burden score and bone mineral density parallel with the improvement in hematological parameters. We assessed the effects of EGT and AMB on GCase activity and autophagy-lysosomal pathway (ALP) in primary cell lines derived from patients with GD2-3 and compared to cell lines from healthy controls. We found that EGT, same as AMB, enhanced GCase activity in control cells and that an individualized response, that varied with GBA mutations, was observed in cells from patients with GD2-3. EGT and AMB enhanced the formation of lysosomal/late endosomal compartments and improved autophagy, independent of GBA mutations. Both AMB and EGT increased mitochondrial mass and density in GD2-3 fibroblasts, suggesting enhancement of mitochondrial function by activating the mitochondrial membrane potential. These results demonstrate that EGT and AMB, with different molecular mechanisms of action, enhance GCase activity and improve autophagy-lysosome dynamics and mitochondrial functions.
Highlights
Control or GD types 2 and 3 (GD2-3) fibroblast lines were treated with increasing concentrations of AMB and EGT for 5 days, and enzyme activity was measured
Our results show that the UGCG inhibitor (EGT) and the pharmacologic chaperone (AMB) increase the residual enzymatic activity in primary cell lines from GD2 patients with L444P/L444P;RecΔ55;RecNCiI, and L444P/D409H mutations
Besides the EGT inhibitory activity and the chaperone activity of AMB, our results reveal that both molecules significantly induce autophagic flux, autophagosome-lysosomal fusion, and increased levels of acidic lysosomes in cells derived from GD2 and GD3 patients
Summary
The red fluorescence of LysoTracker was measured in triplicates using a SpectraMax M2 microplate reader with an excitation wavelength: 577 mm; emission wavelength: 590 nm (Molecular Devices, Sunnyvale, CA, USA). MitoTracker Red CMXRos signal was measured in triplicates using SpectraMax M2 microplate reader with excitation/ emission 577–590, or the fluorescence signal was visualized by fluorescent microscopy (Evos, Hatfield, PA, USA). After five days of treatment with AMB and EGT, fibroblasts were stained with mito-ID membrane potential dye solution in clear-bottom black 96-well tissue culture plates for 30 min. Cells were washed three times with PBS, and the fluorescence signals were visualized by fluorescent microscopy (Evos Digital microscope, Evos, Hatfield, PA, USA). 100 μl of CellTiter-Glo reagent was added directly to the samples, and after 15 min incubation, cells were analyzed by measuring bioluminescence signal in a Genini microplate reader (Molecular Device, San Jose, CA). Statistical analyses were performed using Student’s t-test with 2-tailed distribution and 2-sample equal variance or 1-way ANOVA followed by Student-Newman-Keuls using GraphPad Prism (GraphPad, San Diego, CA, USA)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
